Figure 5.

PPAR-γ agonists and antagonists as well as PPAR-γ KD influence TRPC6 promoter activity, TRPC6 expression, and channel activity. Cultured podocytes were injured by adriamycin (ADRIA) or PAN application and treated with different concentrations of the PPAR-γ agonist (A) pioglitazone or (B) rosiglitazone, and TRPC6 mRNA expression was determined. (C) In addition, TRPC6 expression was determined in uninjured podocytes, transfected with scrambled or PPAR-γ siRNA, and treated with various concentrations of the PPAR-γ antagonist GW9662. A luciferase assay was performed to determine TRPC6 promoter activity in Opossum Kidney cells treated with (D) pioglitazone, (E) rosiglitazone, and (F) GW9662. TRPC6-to-GAPDH and Firefly-to-Renilla ratios were calculated and normalized for vehicle-treated cells. ChIP assays were performed to determine whether PPAR-γ directly binds to the TRPC6 promoter. This was tested using (G) a promoter construct transfected into OK cells or (H) the endogenous TRPC6 promoter in cultured mouse podocytes. Antibodies against histone H3 (H3) as positive control, PPAR-γ to determine whether PPAR-γ bound to the TRPC6 promoter, and IgG as negative control were used to purify fractions of the DNA. (G and H) Hereafter, the proteins were digested, PCR was performed with primers detecting the TRPC6 promoter, and products were put on gel. (I) Western blot analysis showed that PPAR-γ was present in the input sample, in the PPAR-γ–immunoprecipitated sample, and in the H3-positive control sample of the nontransfected MPC-5 cells. Cultured podocytes stably transfected with scrambled siRNA were pretreated with sildenafil or 8-Br-cGMP with or without KT5823 for 24 hours. (J) After removal of the specific media, cells were exposed to 100 μM 1-oleoyl-2-acetyl-sn-glycerolin (OAG) to activate the TRPC6 ion channel. Intracellular Ca²⁺ concentration was determined by fura-2 ratiometry. The arrow indicates OAG application; n=4–8, in at least two independent experiments. Statistical significance was determined using ANOVA followed by (A–F) Bonferroni post hoc test or (J) repeated measurement test. *P<0.05 versus vehicle-treated uninjured cells; ^#P<0.05 versus ADRIA-treated cells; ^$P<0.05 versus PAN-treated cells; ^†P<0.05 versus scrambled transfected uninjured cells; ^¥P<0.05 versus the GW9662 of equal concentration-treated scrambled cells; ^£P<0.05 versus the GW9662 of equal concentration-treated PPAR-γ KD cells; ^±P<0.05 versus the ADRIA-challenged GW9662 of equal concentration-treated cells.